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      Current status of neutron crystallography in structural biology

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          Abstract

          Hydrogen atoms and hydration water molecules in proteins are essential for many biochemical processes, especially enzyme catalysis. Neutron crystallography enables direct observation of hydrogen atoms, and reveals molecular recognition through hydrogen bonding and catalytic reactions involving proton-coupled electron transfer. The use of neutron crystallography is still limited for proteins, but its popularity is increasing owing to an increase in the number of diffractometers for structural biology at neutron facilities and advances in sample preparation. According to the characteristics of the neutrons, monochromatic or quasi-Laue methods and the time-of-flight method are used in nuclear reactors and pulsed spallation sources, respectively, to collect diffraction data. Growing large crystals is an inevitable problem in neutron crystallography for structural biology, but sample deuteration, especially protein perdeuteration, is effective in reducing background levels, which shortens data collection time and decreases the crystal size required. This review also introduces our recent neutron structure analyses of copper amine oxidase and copper-containing nitrite reductase. The neutron structure of copper amine oxidase gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions. Electron transfer via a hydrogen-bond jump and a hydroxide ion ligation in copper-containing nitrite reductase are clarified, and these observations are consistent with the results from the quantum chemical calculations. This review article is an extended version of the Japanese article, Elucidation of Enzymatic Reaction Mechanism by Neutron Crystallography, published in SEIBUTSU-BUTSURI Vol. 61, p.216–222 (2021).

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          Most cited references34

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          Joint X-ray and neutron refinement with phenix.refine.

          Approximately 85% of the structures deposited in the Protein Data Bank have been solved using X-ray crystallography, making it the leading method for three-dimensional structure determination of macromolecules. One of the limitations of the method is that the typical data quality (resolution) does not allow the direct determination of H-atom positions. Most hydrogen positions can be inferred from the positions of other atoms and therefore can be readily included into the structure model as a priori knowledge. However, this may not be the case in biologically active sites of macromolecules, where the presence and position of hydrogen is crucial to the enzymatic mechanism. This makes the application of neutron crystallography in biology particularly important, as H atoms can be clearly located in experimental neutron scattering density maps. Without exception, when a neutron structure is determined the corresponding X-ray structure is also known, making it possible to derive the complete structure using both data sets. Here, the implementation of crystallographic structure-refinement procedures that include both X-ray and neutron data (separate or jointly) in the PHENIX system is described.
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            Biomolecular Deuteration for Neutron Structural Biology and Dynamics.

            Neutron scattering studies provide important information in structural biology that is not accessible using other approaches. The uniqueness of the technique, and its complementarity with X-ray scattering, is greatest when full use is made of deuterium labeling. The ability to produce tailor-made deuterium-labeled biological macromolecules allows neutron studies involving solution scattering, crystallography, reflection, and dynamics to be optimized in a manner that has major impact on the scope, quality, and throughput of work in these areas. Deuteration facilities have now been developed at many neutron centres throughout the world; these are having a crucial effect on neutron studies in the life sciences and on biologically related studies in soft matter. This chapter describes methods that have been developed for the efficient production of deuterium-labeled samples for a wide range of neutron scattering applications. Examples are given that illustrate the use of these samples for each of the main techniques. Perspectives for biological deuterium labeling are discussed in relation to developments at current facilities and those that are planned in the future.
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              Neutron macromolecular crystallography with LADI-III.

              At the Institut Laue-Langevin, a new neutron Laue diffractometer LADI-III has been fully operational since March 2007. LADI-III is dedicated to neutron macromolecular crystallography at medium to high resolution (2.5-1.5 Å) and is used to study key H atoms and water structure in macromolecular structures. An improved detector design and readout system has been incorporated so that a miniaturized reading head located inside the drum scans the image plate. From comparisons of neutron detection efficiency (DQE) with the original LADI-I instrument, the internal transfer of the image plates and readout system provides an approximately threefold gain in neutron detection. The improved performance of LADI-III, coupled with the use of perdeuterated biological samples, now allows the study of biological systems with crystal volumes of 0.1-0.2 mm(3), as illustrated here by the recent studies of type III antifreeze protein (AFP; 7 kDa). As the major bottleneck for neutron macromolecular studies has been the large crystal volumes required, these recent developments have led to an expansion of the field, extending the size and the complexity of the systems that can be studied and reducing the data-collection times required.
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                Author and article information

                Contributors
                tamada.taro@qst.go.jp
                Journal
                Biophys Physicobiol
                Biophys Physicobiol
                Biophysics and Physicobiology
                The Biophysical Society of Japan
                2189-4779
                2022
                1 April 2022
                : 19
                : e190009_1-e190009_10
                Affiliations
                [1 ] Institute for Quantum Life Science, National Institutes for Quantum Science and Technology , Tokai, Ibaraki 319-1106, Japan
                Author notes
                Corresponding author: Taro Tamada, Institute for Quantum Life Science, National Institutes for Quantum Science and Technology, 2-4 Shirakata, Tokaimura, Nakagun, Tokai, Ibaraki 319-1106, Japan. ORCID iD: https://orcid.org/0000-0003-1419-8022, e-mail: tamada.taro@ 123456qst.go.jp

                Edited by Takeshi Murata

                Author information
                https://orcid.org/0000-0003-1419-8022
                Article
                JST.JSTAGE/biophysico/e190009 e190009
                10.2142/biophysico.bppb-v19.0009
                9135615
                35666700
                db54c30c-5e36-4822-9e91-0f20d02b7ee6
                2022 THE BIOPHYSICAL SOCIETY OF JAPAN

                This article is licensed under the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 Inter­national License. To view a copy of this license, visit 
 https://creativecommons.org/licenses/by-nc-sa/4.0/.

                History
                : 8 November 2021
                : 29 March 2022
                Categories
                Review Article (Invited)

                enzyme catalysis,deuteration,diffractometer,copper amine oxidase,copper-containing nitrite reductase

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